CN104089997A - Electrochemical immunosensor, and preparation method and application thereof - Google Patents
Electrochemical immunosensor, and preparation method and application thereof Download PDFInfo
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- CN104089997A CN104089997A CN201410233971.0A CN201410233971A CN104089997A CN 104089997 A CN104089997 A CN 104089997A CN 201410233971 A CN201410233971 A CN 201410233971A CN 104089997 A CN104089997 A CN 104089997A
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Abstract
The invention discloses an electrochemical immunosensor. An antibody for resisting phosphothreonine lyase is at the center of the electrode surface of the electrochemical immunosensor, and is preferably a monoclonal antibody; a sensor preferably contains a double-layer nano-gold membrane; and an electrode of the electrochemical immunosensor is preferably a glass-carbon electrode. The phosphothreonine-lyase electrochemical immunosensor modified by the double-layer nano-gold is constructed by taking chitosan as a bridging agent, fixing a first layer of nano-gold on glass-carbon electrode and adsorbing and fixing the antibody for resisting phosphothreonine lyase, fixing a thionine-chitosan /nano-gold-horseradish peroxidase (HRP) complex to the electrode by dropping onto the electrode, and adsorbing the antibody for resisting phosphothreonine lyase. The electrochemical immunosensor is applicable to detect salmonella and/or shigella in food, is high in sensitivity and strong in specificity, and is capable of realizing rapid quantitative detection on salmonella and/or shigella.
Description
Technical field
The present invention relates to biological immune detection technique field, particularly relate to a kind of electrochemical immunosensor and its preparation method and application; Preferably, described electrochemical immunosensor is threonine phosphoric acid lyase double-layer nanometer gold membrane electrochemical immunosensor.
Background technology
Along with the increasingly extensive application of monoclonal antibody hybridoma technology, need to carry out systematically Analysis and Identification to the monoclonal antibody (McAb) obtaining, to determine its value in various immunological testings.Except the immunoglobulin chain physicochemical property to McAb is analyzed, measure specificity, dissociation equilibrium constant K D, the binding affinity K of McAb conjugated antigen
onand dissociation rate constant K
disvery important for a certain specific purpose to selecting suitable McAb.Bio-molecular interaction instrument adopts biofilm to interfere (Bio-Layer Interferometry, BLI) technology, without mark, has the features such as high sensitivity, high flux, can be used as the instrument of affinity between detectable antigens antibody.Since 2004 come out, at antigen and antibody, done mutually, little molecule and interactions between protein, protein molecular and protein molecular develop rapidly in the aspect such as work and drug screening mutually, receives increasing concern.
Salmonella (Salmonella) is a kind of Gram-negative bacteria that can cause salmonellosis, is one of modal infecting both domestic animals and human type food-borne pathogens.According to statistics, in the kind food posioning of countries in the world, the normal row umber one of salmonellal food poisoning.Shigella (Shigella) is also the pathogenic entero becteria that a class has hyperinfection.According to WHO statistics, have 1.647 hundred million people to infect dysentery, 1.1 ten thousand people are dead, and wherein majority is 5 years old following children.In adult patients, 10～100cfu Shigella just can be caused a disease by infecting enteron aisle, causes inflammatory reaction.At present, the detection method of Salmonella in Food still adopts traditional National Standard Method GB4789.4-2010, in food, the detection method of Shigella adopts traditional National Standard Method GB4789.5-2012, and said method is the generally accepted standard methods in current countries in the world, and qualification result accurately and reliably.But because bacterial classification has thousands of kinds of serotypes, biochemical characteristic is different, make its check program very complicated loaded down with trivial details, during due to detection, need to repeatedly carry out microbe growth, therefore at least need 4～7d just can obtain testing result accurately.In recent years, detection for salmonella, Shigella has both at home and abroad had some new progresses, be mainly based on optical surface plasma resonance (Surface Plasmon Resonance, SPR) technology, biofilm are interfered the method for quick of (BioLayer Interferometry, BLI) technology, round pcr, fluorescence labeling technology and Enzyme-multiplied immune technique (ELISA) etc.But PCR, SPR, BLI instrument and equipment precision prescribed are high, expensive, can not realize online detection, and test operation personnel are required strictly; And fluorescence labeling technology and ELISA can only carry out qualitative or sxemiquantitative, add data processing and add the restriction of sample loading mode, it is almost impossible realizing instant, accurate quantitative analysis and online detection.Electrochemical analysis has and is not subject to the turbidity of sample, the impact of color, the high efficiency of the relatively simple feature of equipment needed thereby instrument and enzymatic reaction, immunoassay, without sample being carried out to the pretreated features such as purifying, enrichment, can better become the detection platform of salmonella and shigella.
Threonine phosphoric acid lyase (phosphothreonine lyase) be take SpvC (Salmonella plasmid virulence C) and (is comprised Outer shigella protein F as representative, OspF) the enzyme effect protein family that a class is new, by salmonella and shigella, by III type excretory system specific secretion, therefore can be used as the mark that detects two kinds of bacterium.When it is injected into after host cell, irreversible deactivation host MAPK (mitogen-activated protein kinases, MAPKs), destroys host's congenital immunity defence line, the acute inflammation causing.Research is found causing the bacterial classification of bacillary dysentery to secrete SpvC by III type excretory system.The present invention intends take threonine phosphoric acid lyase and prepares anti-SpvC monoclonal antibody as antigen, utilizes specific binding effect between antigen-antibody to detect threonine phosphoric acid lyase, thereby indirectly reaches the object that detects salmonella and shigella; The present invention intends filtering out the anti-SpvC monoclonal antibody strain stronger with antigen protein affinity for the preparation of immunosensor by BSI.
Summary of the invention
The object of the invention is for solving existing defect in prior art the preparation method of a kind of highly sensitive, the high specificity of invention, the electrochemical immunosensor of quantitative determination salmonella and shigella.
For achieving the above object, on the one hand, the invention provides a kind of electrochemical immunosensor, there is the antibody of anti-threonine phosphoric acid lyase at the center of the electrode surface of described sensor.
Described antibody is polyclonal antibody.
Described antibody is monoclonal antibody.
Described sensor contains double-layer nanometer gold film.
Described electrode is glass-carbon electrode.
Described sensor take shitosan as bridging agent fixedly ground floor nm of gold in the antibody of glass-carbon electrode the fixing anti-threonine phosphoric acid lyase of absorption.
Described sensor is fixed on electrode in dropping electrode mode by thionine-shitosan/nm of gold-horseradish peroxidase (HRP) compound, and adsorb the antibody of anti-threonine phosphoric acid lyase, build the threonine phosphoric acid lyase electrochemical immunosensor that double-layer nanometer gold is modified.
On the one hand, the preparation method of the electrochemical immunosensor described in the present invention also provides, comprises the steps:
1) preparation of the antibody of anti-threonine phosphoric acid lyase;
2) pre-service of glass-carbon electrode;
3) preparation of nano gold sol;
4) preparation of thionine/shitosan (Thi/Chit) multipolymer;
5) preparation of nm of gold absorption horseradish peroxidase;
6) preparation of threonine phosphoric acid lyase double-layer nanometer gold membrane electrochemical immunosensor.
Described antibody is polyclonal antibody or monoclonal antibody.
On the one hand, the present invention also provides described electrochemical immunosensor to detect the detection method of Salmonella in Food and/or Shigella.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention take shitosan as bridging agent fixedly ground floor nm of gold in the antibody of glass-carbon electrode the fixing anti-threonine phosphoric acid lyase of absorption; In dropping electrode mode, thionine-shitosan/nm of gold-horseradish peroxidase (HRP) compound is fixed on to above-mentioned electrode, and adsorb the antibody (the preferred monoclonal antibody of described antibody) of anti-threonine phosphoric acid lyase, built the threonine phosphoric acid lyase electrochemical immunosensor that double-layer nanometer gold is modified, good signal amplifying system in this formation makes it highly sensitive, high specificity, can realize Quantitative detection to salmonella and/or Shigella.
Accompanying drawing explanation
Fig. 1 is nano gold sol electromicroscopic photograph of the present invention, and upper left corner scale is 50nm, and upper right corner scale is 20nm, and lower left corner scale is 10nm, and lower right corner scale is 5nm.
Fig. 2 is the schematic diagram of preparing of threonine phosphoric acid lyase double-layer nanometer gold membrane electrochemical immunosensor of the present invention.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
The preparation of embodiment 1 sensor
1, the screening of anti-threonine phosphoric acid lyase monoclonal antibody strain
First, adopt conventional method for preparing monoclonal antibody, by deriving from the SpvC proteantigen injection Balb/C mouse of salmonella, obtain monoclonal antibody strain, then lumbar injection Balb/C mouse, purifying ascites obtain anti-threonine phosphoric acid lyase monoclonal antibody.
Then, carry out the screening of anti-threonine phosphoric acid lyase monoclonal antibody strain: the bio-molecular interaction instrument Octet Red96 that adopts U.S. ForteBio company to produce carry out this experiment: get five Protein A sensors A 3, B3, C3, D3, E3, five concentration gradient threonine phosphoric acid lyase proteantigens are corresponding one by one with it, concentration is respectively: 4 μ M, 1.33 μ M, 0.44 μ M, 0.1481 μ M, 0 μ M.Five strain SpvC monoclonal antibodies used are respectively: A2-E2 (1.129 μ M); A3-E3 (0.91 μ M); A4-E4 (0.91 μ M); A5-E5 (1.755 μ M); A6-E6 (1 μ M) result is as following table.
Dissociation equilibrium constant, binding constant and the dissociation constant of the strain of table 1 different antibodies
The dissociation equilibrium constant K D (KD=kdis/kon) of the anti-SpvC monoclonal antibody of A2-E2 strain and SpvC as seen from the above table, minimum is 3.85E-10M, and affinity is the strongest, and this strain monoclonal antibody can be for the preparation of following immunosensor.And five strain monoclonal antibody binding constant kon values are 2.50E+031/Ms, show that antigen protein equates with five strain monoclonal antibody binding abilities, in 0.91 μ M～1.755 μ M concentration range endoantigen and monoclonal antibody binding ability and concentration, have nothing to do, its affinity size depends on dissociation constant kdis.
The polyclonal antibody that it will be appreciated by persons skilled in the art that anti-SpvC also can be for the preparation of electrochemical immunosensor of the present invention.
2, the pre-service of glass-carbon electrode:
Glass-carbon electrode is used respectively successively to the α-Al of 1.0 μ m, 0.3 μ m, 0.05 μ m particle diameter
2o
3slurry polishing three times on chamois leather, and in ultrasonic water bath, clean 30s after each polishing, finally use successively HNO
3with H
2mixed liquor, absolute ethyl alcohol, ultrapure water that O is configuration in 1: 1 according to volume ratio clean.At 1mol/L H
2sO
4in solution, by sweep limit, be 1.0～-1.0V, the cyclic voltammetry activated electrode that sweep velocity is 100mV/s, until there is stable cyclic voltammetry curve in multiple scanning.Above-mentioned stable cyclic voltammetry curve meets following requirement: under laboratory condition, the spike potential of the cyclic voltammetry curve of pretreated electrode is poor should be below 80mV, and approach as much as possible 64mV, electrode can be used, and is finally placed in nitrogen environment dried for standby.
3, the preparation of nm of gold (Nano-Au) colloidal sol:
According to Frens method, get 0.01g/100mL chlorauric acid solution 100mL, add the sodium citrate solution 4mL of 1g/100mL to mix, be placed in the low fire of micro-wave oven and keep 8-10min, after it is naturally cooling, with ultrapure water, be supplemented to 104mL and obtain nano gold sol, be placed in 4 ℃ and keep in Dark Place standby.Utilize spectrophotometer to scan prepared nano gold sol (400-700nm) in visible-range, light absorption collaurum has a single optical absorption peak in visible-range, and the wavelength X of optical absorption peak
maxat 518nm, can determine roughly that nm of gold mean grain size is 15nm.With transmission electron microscope, the size of made nano gold sol particle, shape and deployment conditions are carried out to further accurate sign, the nm of gold regular shape of synthesized, epigranular, mean grain size is about 15nm, and there is no clustering phenomena.
The electromicroscopic photograph of nano gold sol is shown in Fig. 1.
4, the preparation of thionine/shitosan (Thi/Chit) multipolymer:
(1) 2g shitosan (Chit) being dissolved in to 100mL percent by volume is in 2% acetum, to stir 3h to obtain 2% chitosan solution.
(2) chitosan solution that is 2% by above-mentioned 2.5mL quality percent by volume is added in the glutaraldehyde solution that 320 μ L percents by volume are 10%, after mixing, add 0.01M thionine (Thi) solution 200 μ L, finally adding percent by volume is that 2% acetum to cumulative volume is 6mL, after mixing, can be used for dripping painting electrode, and this copolymer solution needs matching while using.
5, the preparation of nm of gold absorption horseradish peroxidase:
(1) 0.02g horseradish peroxidase (HRP) is dissolved in the phosphate buffer (PBS) that 0.01M pH value is 7.4 to configuration 2.0g/L horseradish peroxidase solution.
(2) use 0.1M K
2cO
3after the pH value to 7.0 of the nano gold sol of regulating step 2 preparations, getting nano gold sol and the above-mentioned 2.0g/L horseradish peroxidase of the 1mL solution stirring 2h that the above-mentioned pH value of 1mL is 7.0 mixes, 4 ℃ of standing over night, and utilize spectrophotometer to scan within the scope of 350～700nm this mixed liquor, obtain the absorption spectrum of HRP/Nano-Au polymkeric substance, by maximum absorption wavelength, Nano-Au and the rear situation of change occurring of HRP interaction are characterized.
6, the preparation of threonine phosphoric acid lyase double-layer nanometer gold membrane electrochemical immunosensor:
(1) 0.5g shitosan being dissolved in to 100mL volume fraction is 1% acetum, and obtaining quality percent by volume is 0.5% chitosan solution.
(2) getting the chitosan solution that the above-mentioned quality percent by volume of 5 μ L is 0.5% drips in the pretreated glass-carbon electrode of step 1 surface, be placed in the dry 20min of 50 ℃ of baking ovens, make described chitosan solution form the gel that encases glass-carbon electrode, taking-up is dipped in 5min in 1mol/L NaOH solution after it naturally cools to room temperature, with ultrapure water, clean rear and be dipped in 30min in ultrapure water, NaOH is all entered in water.
(3) take out, at the dry 10min of 25 ℃ of drying boxes; Then be placed at least 24h of nano gold sol, obtain nm of gold electrode.Then the 4 ℃ of self assemblies of anti-threonine phosphoric acid lyase monoclonal antibody that this electrode are placed in to purifying are 24h at least, obtains the electrode of individual layer decorated by nano-gold.
(4) getting thionine/shitosan (Thi/Chit) copolymer solution prepared in 5 μ L steps 4 drips in the center of the electrode surface of individual layer decorated by nano-gold again, natural drying rear formation polymer film, with ultrapure water repeatedly clean formed polymer film to wash out water under 600nm without light absorption value, then be placed at least 24h of the prepared 4 ℃ of self assemblies of nm of gold/horseradish peroxidase solution of step 5, ultrapure water is placed at least 24h of the purified 4 ℃ of self assemblies of threonine phosphoric acid lyase monoclonal antibody of step 3 after rinsing again, bovine serum albumin solution (BSA) solution that is finally placed in 1g/100mL is in 37 ℃ of incubations 1h at least, to seal non-specific site, to take containing percent by volume be 0.05%Tween-20, and phosphate buffer cleans unconjugated bovine serum albumin(BSA), naturally dry and obtain the threonine phosphoric acid lyase electrochemical immunosensor of double-layer nanometer gold modification.This sensor can be stored in 4 ℃ of PBS damping fluids stand-by.
Preparation and the detection principle of threonine phosphoric acid lyase double-layer nanometer gold membrane electrochemical immunosensor of the present invention are shown in Fig. 2.Can utilize cyclic voltammetry, AC impedence method, atomic force microscope etc. to characterize each stage of electrode assembling.
Embodiment 2 sensors detect salmonella and/or Shigella
1, preparation standard curve:
By the threonine phosphoric acid lyase double-layer nanometer gold membrane electrochemical immunosensor preparing respectively in the salmonella suspension of 0.01M pH7.4PBS damping fluid gradient dilution 37 ℃ carry out electric current-time scan (scanning current potential-0.38V) after hatching 15min (from low concentration to high concentration), through alternative steady-state current value (being generally 50s), it is standard, variable quantity △ I with electric current before and after detecting and Salmonella bacterium colony number (measure > > according to GB4789.2 < < microbiological test of food hygiene total plate count and measure bacteria concentration) mapping, obtain the linear relationship between △ I and salmonella concentration.
2, sample preparation:
Select concentration to be about 1 * 10
8cfu/mL salmonella suspension (detect and obtain according to GB4789.2), therefrom draws 1mL and joins in 9mLPBS damping fluid, and piping and druming shakes up, and is designated as 1, and now bacteria concentration is about 1 * 10
7cfu/mL; From 1, draw 1mL solution again and be added drop-wise in 9mLPBS damping fluid, piping and druming shakes up, and is designated as 2, and now bacteria concentration is about 1 * 10
7cfu/mL; The like, until be diluted to 1cfu/mL bacterium solution.Shigella is also cooked corresponding processing, prepares the gradient dilution of mixed bacteria liquid simultaneously.Using PBS damping fluid as blank, and detect successively to high concentration from low concentration.
3, testing result:
Utilize current-time curvel method to measure respectively salmonella and the Shigella suspension of PBS dilution, result shows that response current and two kinds of bacterium are 10～1.0 * 10
4cfu/mL scope internal linear is relevant, and lowest detection is limited to 5cfu/mL.Recycling time current curve method is measured two kinds of bacterium mixed liquors of PBS dilution, and response current and Mixed Microbes are 10～1.0 * 10
4within the scope of cfu/mL, be also linear dependence, show that this sensor can detect bacterium total concentration in these two kinds of mixed bacteria liquids.
Embodiment 3 sensor stabilities and reappearance detect
By the threonine phosphoric acid lyase double-layer nanometer gold membrane electrochemical immunosensor assembling respectively 1 * 10
2cfu/mL salmonella and 1 * 10
2in the PBS dilution of cfu/mL Shigella, METHOD FOR CONTINUOUS DETERMINATION is 12 times, and result relative standard deviation R.S.D is respectively 3.23%, 4.11%; This sensor, in the 4 ℃ of preservations in top of 0.01mol/L pH7.4PBS solution, was detected to a salmonella suspension (1 * 10 every 3 days
2cfu/mL), the 13rd day current responsing signal is initial current 86.45%, shows that this sensor has good stability.
Get 5 of immunosensors prepared by different batches, under the same conditions to same concentration salmonella suspension (1 * 10
2cfu/mL), Shigella suspension (1 * 10
2cfu/mL) and two kinds of mixed bacteria liquids (1 * 10
2cfu/mL) measure, the relative standard deviation R.S.D. of result response current is respectively 4.12%, 4.66%, 4.82%, illustrates that this sensor reappearance is good.
Embodiment 4 sensor disturbance tests
Use respectively 1 * 10
7the bacillus subtilis of cfu/mL, Escherichia coli, staphylococcus aureus, Sarcina lutea have carried out interference test, and result shows this sensor quantitative measurement salmonella and shigella (1 * 10
7cfu/mL) all without obviously disturbing.
Above embodiment shows, sensor combined with electrochemical of the present invention analysis can not be subject to the impact of sample turbidity, color, equipment needed thereby instrument is relatively simple, without sample being carried out to the pre-service such as purifying, enrichment, can be applicable to respectively or the fast detecting to salmonella and shigella simultaneously.
Although the present invention is described in detail above to have used general explanation and specific embodiment, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. an electrochemical immunosensor, is characterized in that, there is the antibody of anti-threonine phosphoric acid lyase at the center of the electrode surface of described sensor.
2. electrochemical immunosensor according to claim 1, is characterized in that, described antibody is polyclonal antibody.
3. electrochemical immunosensor according to claim 1, is characterized in that, described antibody is monoclonal antibody.
4. according to the electrochemical immunosensor described in claim 1-3 any one, it is characterized in that, described sensor contains double-layer nanometer gold film.
5. according to the electrochemical immunosensor of aforementioned any one, it is characterized in that, its electrode is glass-carbon electrode.
6. according to the electrochemical immunosensor of aforementioned any one, it is characterized in that, take shitosan as bridging agent fixedly ground floor nm of gold in the antibody of glass-carbon electrode the fixing anti-threonine phosphoric acid lyase of absorption.
7. according to the electrochemical immunosensor of claim 3, it is characterized in that, in dropping electrode mode, thionine-shitosan/nm of gold-horseradish peroxidase (HRP) compound is fixed on to electrode, and adsorb the antibody of anti-threonine phosphoric acid lyase, build the threonine phosphoric acid lyase electrochemical immunosensor that double-layer nanometer gold is modified.
8. the preparation method of the electrochemical immunosensor described in aforementioned any one claim, comprises the steps:
1) preparation of the antibody of anti-threonine phosphoric acid lyase;
2) pre-service of glass-carbon electrode;
3) preparation of nano gold sol;
4) preparation of thionine/shitosan (Thi/Chit) multipolymer;
5) preparation of nm of gold absorption horseradish peroxidase;
6) preparation of threonine phosphoric acid lyase double-layer nanometer gold membrane electrochemical immunosensor.
9. preparation method according to Claim 8, wherein, described antibody is polyclonal antibody or monoclonal antibody.
10. the electrochemical immunosensor described in aforementioned any one claim detects the detection method of Salmonella in Food and/or Shigella, it is characterized in that, by described sensor for detection of food.
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| CN105277604A (en) * | 2015-11-10 | 2016-01-27 | 北京盈盛恒泰科技有限责任公司 | Electrochemical nanometer immunosensor for cannabinol detection and preparation method and application thereof |
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| CN110632301A (en) * | 2019-09-25 | 2019-12-31 | 吉林大学 | Rapid salmonella detection method based on biofilm interference technology |
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